Peripheral neuropathic pain results from maladaptive changes in the central nervous system that are initiated by abnormal activity of injured sensory neurons. Increasing evidence indicates that neuroplasticity in the spinal cord contributes to the development and maintenance of neuropathic pain. Our published and preliminary data indicate that peptides derived from the neurosecretory protein VGF (non-acronymic) may function as sensory neuron signals that initiate and maintain sensitization of spinal neurons after nerve injury [3]. The proposed studies will focus on the spinal signaling mechanisms of the VGF-derived peptide TLQP-21 and will build upon the identification this year of C3aR1 (complement 3a receptor) as a receptor for TLQP-21. Our preliminary data show that endogenous TLQP-21 contributes to nerve injury-induced hypersensitivity and that TLQP-21 induced hyperalgesia is blocked by C3aR1 inhibition. These findings implicate C3aR1 as a novel therapeutic target for the inhibition of spinal neuropathic pain transmission. The objective of this application is to establish the TLQP21/C3aR1 system as an essential functional component of neuropathic pain. The central hypothesis of this proposal is that TLQP-21 activation of C3aR1 in dorsal horn of spinal cord establishes and maintains neuropathic pain. Specific aim 1 will test the hypothesis that the spinal effects of TLQP-21 are mediated by C3aR1 and that C3aR1 is involved in the establishment of neuropathic pain. The proposed studies will (1) characterize pharmacologically the ligand-receptor relationship of TLQP-21 and C3aR1 in spinal cord, (2) determine whether the spinal effects of TLQP-21 are dependent on C3aR1 activation, and (3) determine whether C3aR1 contributes to behavioral signs of neuropathic pain. Specific aim 2 will test the hypothesis that nerve injury increases expression of C3aR1 in neurons, microglia, and/or astrocytes. These studies will examine the effects of nerve injury on spinal C3aR1 using (1) expression analysis, and (2) antibody- independent visualization of TLQP-21/C3aR1 binding in dorsal horn. Specific aim 3 will address the hypothesis that neurophysiological responses to TLQP-21/C3aR1 activation in dorsal horn are enhanced after nerve injury. These studies will examine the effects of nerve injury on (1) TLQP-21 modulation of neuronal activity in dorsal horn using in vivo extracellular recording, and (2) TLQP-21 evoked Ca2+ transients in spinal cord slices. At the completion of these studies we will have determined the relationship of TLQP-21 to C3aR1 and their role in the development and maintenance of neuropathic pain. Such information will have a significant impact because it will define the TLQP-21/C3aR1 as a potential pharmacotherapeutic target for neuropathic pain. The knowledge acquired from this research will greatly advance the field of chronic pain and may extend to other therapeutic areas related to neuroplasticity.